The subfamily Bambusoideae (Poaceae) comprises both woody and herbaceous bamboos, but only woody bamboos have economic potential. At present about 120 genera of temperate and tropical woody bamboos are recognized, which are distributed worldwide covering 18 million hectares (ha), mainly in South East Asia, with about 3.8 million hectares in China and 8 million ha (13.8% of the forest land) in India.
Bamboos are versatile plants with many different applications. It has been estimated that about 2.2 billion people worldwide use bamboo more or less frequently and in 1985 the global revenue of bamboo was estimated around U.S. $4.5 billion. But bamboo also has a great future potential, since the use of bamboo as a raw industrial material is becoming more and more important and an increasing number of products are exported. Europe and North America import a lot of bamboo material from Asia such as toothpicks, sate sticks, brooms, poles for viticulture and arboriculture, small bamboo sticks for the production of ornamental plants and vegetables. Moreover bamboo parquet, new laminated products and other wood industry products, as well as exotic furniture, and a variety of handicraft items are found on the retail market.
The demand for bamboos will be exceedingly high, mainly due to serious forest depletion and the possibilities of using bamboo as a substitute for tropical timber. Currently the existing propagation techniques are clearly insufficient to meet the projected demands. While many classical techniques, such as clump division and cutting, are widely practiced, both their capacity to produce mass scale production, and their practical efficiency, are far too low. For example, to produce cutting, complete culms can be buried below soil level for some species. The rate of success is typically only 50% and for the production of 100,000 plants 10 hectares are needed.
The search for one single method for large scale production for all bamboos remains thus highly desirable. Micropropagation is an excellent method to achieve this aim. In a short period of time large numbers of bamboos can be produced in the laboratory, starting from elite selected genotypes, which can be transported by air easily to any place in the world. Moreover tissue cultured plants are generally very vigorous growers and disease free.
Within micropropagation it is necessary to distinguish between large scale production and mass scale production. Large scale production typically ranges between 10,000 and 1 million plants per species per annum. A production of this size suffices for production as ornamental or for reforestation programs of the order of 2000 hectares. Especially in ornamental production and in pilot scale plantation, the added value is sufficiently high to use the method of axillary branching in vitro. This method has the additional advantages that of all possible methods for micropropagation, it has the lowest chance for genetic aberration, which certainly is of prime importance for ornamental production and pilot scale plantations, with feedback times for genotypic identity of 5-10 years at minimum.
Mass scale production on the other hand, aims at producing bamboos at the mass scales demanded by reforestation schemes. Magnitudes are in the order of 10-100 million plants per annum. The production of propagules for field planting and establishment has to be very cost-effective (read prices are very low) and the propagules cannot be handled and planted individually. The micropropagation method of choice here is somatic embryogenesis and encapsulation of these embryos into synthetic seeds. This allows the actual seeding of millions of plants.
Research papers have been published both on axillary branching and somatic embryogenesis of bamboo, but transforming the published protocols into practical economic propagation has been successful in only a very limited number of cases. Although there is published literature concerning tissue culture of bamboo, each paper tends to present a method applicable to only a single species. In the United States, at present all commercial bamboo is from vegetative culture and not from tissue culture.
The present proposal presents an invention which allows one to micropropagate any type of bamboo at both large and mass scale. It circumvents the problems encountered in research papers and in transforming research protocols into economically viable propagation systems. The economic viability of micropropagation systems is based on the large or mass scale production of high quality plants.
The added value of the tissue culture process can be maximized by using elite planting material that is selected on the basis of well defined selection criteria. Given the long feedback time for evaluation of genotypes and the location of the laboratory in relation to the habitat of the genotype (which may be several 1000 km apart), it is advisable to develop suitable storage methods. The suitability of tissue culture plants or propagules, provides excellent material for low temperature storage or cryopreservation. In this invention these methods are included as well.
The difficulties of propagation as currently practiced will be outlined followed by a description of the invention including a schematic diagram explaining the invention.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
The invention consists of several novel solutions to overcome specific problems currently encountered in propagating bamboo. The invention consists of these novel solutions both individually and in combination. The problem areas addressed by the various aspects of the invention include the following:
a) Specific preparation of plants under stage 0 conditions.
b) Use of diseased parts of bamboos as explants for initiation.
c) Reversion of in vivo flowering in vitro.
d) Subculture of flowering parts in vitro as monocultures.
e) Induction of nodule cultures in monocultures of flowering parts.
f) Induction of flowering in vitro by specific combination of chemical and physical parameters.
g) Induction of somatic embryogenesis in adult bamboos without prior organogenesis.
h) Production of synthetic seeds.
i) Dehydration of somatic embryos prior to the production of synthetic seeds.
j) Use of somatic embryos, nodule cultures and shoot tips for cryopreservation using an encapsulation dehydration method.
k) Use of bottomcooling and increased light intensity prior to transplantation.
l) Use of micropropagated plants as micromotherplants for further multiplication.
m) The use of an acetone rinse for sterilization.
The details for performing each of the above are set out in the Examples. The invention teaches how to begin with seeds, vegetative mature seedlings or inflorescences and efficiently micropropagate these on a large or mass scale. A variety of conditions including type of medium, concentrations of auxin and cytokinins, temperature, lighting, humidity and sterilization are disclosed, the use of which results in a high percentage success rate of micropropagation thereby allowing economically viable production of bamboo on a large or mass scale. One or more pathways shown in FIG. 1 are applicable to all species of bamboo including woody tropical, woody temperate and herbaceous species.